<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), a major <em>factor</em> mediating <em>endothelial</em> cell survival, migration, and proliferation during angiogenesis, is expressed as five splice variants (<em>121</em>, 145, 165, 189, and 206 aminoacids) encoded by a single gene. Although the three shorter isoforms are mainly diffusible, the two longer ones are sequestered in cell membranes after secretion. However, their potential role as true components of the extracellular matrix has not been investigated. We determined that <em>endothelial</em> cells could adhere and spread on VEGF189 and VEGF165, but not on VEGF<em>121</em>. Adhesion was mediated by the alpha3beta1 and alpha(v)beta3 integrins and other alpha(v) integrins but not by the cognate VEGF receptors. Cells migrated on VEGF165 and VEGF189 and displayed a stellate morphology with numerous lamellopodia and FAK staining but no actin stress fibers. Tumstatin, an antiangiogenic peptide that interacts with the alpha(v)beta3 integrin, could inhibit adhesion on VEGF, and this effect was potentiated by anti-alpha(v)beta3 blocking antibody. Immobilized VEGF almost totally abolished <em>endothelial</em> cell apoptosis through interactions with integrins. The inhibition of alpha(v)beta3 engagement with immobilized VEGF by tumstatin inhibited most of its survival activity. We have thus determined a new VEGF receptor-independent role for immobilized VEGF in supporting cell adhesion and survival through interactions with integrins.

Angiogenesis is a process of development and of <em>growth</em> of new capillary blood vessels from pre-existing vessels. When pathological, it contributes to the development of numerous types of tumors, and the formation of metastases. In order to grow, carcinoma need new blood vessels to form so that they can feed themselves. Therefore, nowadays the concept according to which the development of cancer is angiogenesis dependent is generally recognized. This concept makes the control of tumoral angiogenesis one of the promising therapeutic ways in cancerology. The transition from the latent phase to the invasive and metastatic phase of a cancer is linked to what is called the angiogenic switch. It implies complex cellular and molecular interactions between cancerous cells, <em>endothelial</em> cells and the components of the extra-cellular matrix and namely the existence of specific proteins secreted by the tumoral cells able to stimulate the proliferation of capillary <em>endothelial</em> cells. Among them, VEGF, <em>Vascular</em> <em>Endothelial</em> <em>Growth</em> <em>Factor</em> was found in several types of tumors. It has shown a tumoral angiogenic activity in vitro and in vivo, and thus is a privileged target for the control of angiogenesis in an anti-tumoral goal. The role of VEGF in tumoral angiogenesis has been extensively studied. It has been proved to undergo as well autocrine as paracrine stimulation of tumoral angiogenesis. During the last few years, several members of the VEGF family have been described namely the VEGF-A, B, C, D, E and placenta <em>growth</em> <em>factor</em> (PlGF) among which VEGF-A (<em>121</em> aminoacids) plays a role of prime importance in angiogenesis. VEGF is a 45 kDA glycoprotein, homodimeric, basic, and able to bind heparin. The three-dimensional structure of VEGF has been recently determined, by X-rays diffraction, and NMR spectroscopy. The different forms of the VEGF bind to receptors that exhibit a tyrosine-kinase activity (RTK). The specific action of the VEGF on the <em>endothelial</em> cells is mainly regulated by two types of RTK of the VEGF family, VEGFR1, or Flt-1, and VEGFR2, or KDR/Flk-1. Mutagenesis studies have shown that only a small number of VEGF residues are important and essential for the binding with RTK. Data described to date from the studies of VEGF/RTK interactions agree to the hypothesis that KDR receptor is the main human receptor responsible for the VEGF activity in both physiological and pathological <em>vascular</em> development, and VEGF-KDR signalling pathway has been validated as a priority target for the development of anti- and pro- angiogenic agents. Therefore angiogenesis mediated by VEGF constitutes a new target for anti-cancer therapy which has explored through different ways of intervention aiming at the blocking of the tumoral angiogenesis. The main ones are: -Struggle against the stroma degradation and invasion by the neo-vessels -Inhibition of activated <em>endothelial</em> cells. -Inhibition of angiogenic <em>factors</em> production and of their receptors. -Inhibition of the VEGF signal pathway, by peptides blocking the bond between VEGF and its receptors through the inhibition of intracellular transduction of VEGF signal. In conclusion, this bibliographic study allows to situate works of medicinal chemistry in the context of present knowledge concerning the <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) and its role in angiogenesis.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is a major agent in choroidal and retinal neovascularization, events associated with age-related macular degeneration (AMD) and diabetic retinopathy. Retinal pigment epithelium (RPE), strategically located between retina and choroid, plays a critical role in retinal disorders. We have examined the effects of various <em>growth</em> <em>factors</em> on the expression and secretion of VEGF by human retinal pigment epithelial cell cultures (HRPE). RT-PCR analyses revealed the presence of three isoforms of mRNA corresponding to VEGF <em>121</em>, 165, and 189 that were up regulated by TGF-beta1. TGF-beta1, beta2, and beta3 were the potent inducers of VEGF secretion by HRPE cells whereas bFGF, PDGF, TGF-alpha, and GM-CSF had no effects. TGF-beta receptor type II antibody significantly reversed induction of VEGF secretion by TGF-beta. In contrast activin, inhibin and BMP, members of TGF-beta super family, had no effects on VEGF expression in HRPE. VEGF mRNA levels and protein secretion induced by TGF-beta were significantly inhibited by SB203580 and U0126, inhibitors of MAP kinases, but not by staurosporine and PDTC, protein kinase C and NF-kappaB pathway inhibitors, respectively. TGF-beta also induced VEGF expression by fibroblasts derived from human choroid of eye. TGF-beta induction of VEGF secretion by RPE and choroid cells may play a significant role in choroidal neovascularization (CNV) in AMD. Since the secretion of VEGF by HRPE is regulated by MAP kinase pathways, MAP kinase inhibitors may have potential use as therapeutic agents for CNV in AMD.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF, VEGF-A), a selective mitogen for <em>endothelial</em> cells is a critical <em>factor</em> for <em>vascular</em> development. Two isoforms that differ in the presence of exons 6 and 7, Vegf(165) and Vegf(<em>121</em>), are the dominant forms expressed in zebrafish embryo. Simultaneous overexpression of both isoforms in the embryo results in increased production of flk1, tie1, scl, and gata1 transcripts, indicating a stimulation of both <em>endothelial</em> and hematopoietic lineages. We also demonstrate that vegf can stimulate hematopoiesis in zebrafish by promoting the formation of terminally differentiated red blood cells. Simultaneous overexpression of both isoforms also causes ectopic vasculature and blood cells in many of the injected embryos as well as pericardial edema in later stage embryos. Overexpression of vegf also resulted in earlier onset of flk1, tie1, scl, and gata1 expression in the embryo, indicating a possible role of vegf in stimulating the differentiation of both <em>vascular</em> and hematopoietic lineages. Co-injection of RNAs for both isoforms results in increased expression of three of these markers over and above that observed when either RNA is singly injected and analysis of vegf expression in the notochord mutants no tail and floating head suggests that the notochord patterns the formation of the dorsal aorta by stimulating adjacent somite cells to express vegf, which in turn functions as a signal in dorsal aorta patterning. Finally, studies of vegf expression in cloche mutant indicate that vegf expression is generally independent of cloche function. These results show that in the zebrafish embryo, vegf can not only stimulate <em>endothelial</em> cell differentiation but also hematopoiesis. Moreover, these effects are most dramatic when both vegf isoforms are co-expressed, indicating a synergistic effect of the expression of the two forms of the VEGF protein.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is expressed by renal glomerular epithelial cells (podocytes) and is thought to be protective against nephrotoxic agents. VEGF has been shown to be an autocrine survival <em>factor</em> in neuropilin-1-positive, VEGF receptor-negative breast carcinoma cells. Normal human podocytes are also known to express neuropilin-1, VEGF, and are VEGF-R2 negative. Here, we investigated whether a similar VEGF autocrine loop may exist in podocytes. Podocyte cytosolic calcium concentration ([Ca(2+)](i)) was analyzed in primary cultured and conditionally immortalized podocytes using ratiometric fluorescence measurement. Cytotoxicity was determined by lactate dehydrogenase assay, proliferation by [(3)H]-thymidine incorporation, and cell counts by hemocytometric assay. VEGF decreased [Ca(2+)](i) in primary podocytes (from 179 +/- 36 to <em>121</em> +/- 25 nM, P < 0.05) and conditionally immortalized podocytes (from 95 +/- 10 to 66 +/- 8 nM, P < 0.02) in the absence of extracellular calcium. The type III receptor tyrosine-kinase inhibitor PTK787/ZK222584 abolished this reduction. VEGF increased podocyte [(3)H]-thymidine incorporation (3,349 +/- 283 cpm, control 2,364 +/- 301 cpm, P < 0.05) and cell number (4.5 +/- 0.7 x 10(4)/ml, control 2.6 +/- 0.5 x 10(4)/ml, P < 0.05) and decreased cytotoxicity (5.9 +/- 0.7%, control 12 +/- 3%, P < 0.05), whereas a monoclonal antibody to VEGF increased cytotoxicity. Electron microscopy of normal human glomeruli demonstrated that the glomerular VEGF is mostly podocyte cell membrane associated. These results indicate that one of the functions of VEGF secreted from podocytes may be to act as an autocrine <em>factor</em> on calcium homeostasis and cell survival.

Assessment of angiogenesis may yield important information for an effective antiangiogenic treatment for hepatocellular carcinoma (HCC) because HCC is characteristically hyper<em>vascular</em> We examined the relationship of microvessel density (MVD), <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), and VEGF receptors Flt-1 and Flk-1/KDR in 50 patients with HCC and in 3 hepatoma cell lines. VEGF messenger RNA (mRNA) was overexpressed in 26 tumors (52%), and the 3 VEGF isoforms (<em>121</em>, 165, and 189) were present in high frequencies. Flt-1 mRNA was overexpressed in 34 tumors (68%), with levels significantly increased in HCCs compared with the nontumorous livers. Tumor Flt-1 mRNA significantly correlated with tumor VEGF mRNA levels. Within the group of tumors 8.5 cm or less in diameter, tumors with intrahepatic metastasis in the form of tumor microsatellite formation had significantly higher VEGF mRNA levels. MVD assessed by immunohistochemical analysis with CD34 antibody was inversely related to tumor size. Angiogenesis as assessed by MVD and tumor VEGF expression seems to have a more important role in tumor <em>growth</em> and intrahepatic metastasis in smaller HCCs. The differential up-regulation of Flt-1 suggests that it may have an important role in angiogenesis in HCC.

OBJECTIVE

Noninvasive positron emission tomography (PET) imaging of vascular endothelial growth factor receptor 2 (VEGFR-2) expression could be a valuable tool for evaluation of patients with a variety of malignancies, and particularly for monitoring those undergoing antiangiogenic therapies that block VEGF/VEGFR-2 function. The aim of this study was to develop a VEGFR-2-specific PET tracer.

METHODS

The D63AE64AE67A mutant of VEGF(121) (VEGF(DEE)) was generated by recombinant DNA technology. VEGF(121) and VEGF(DEE) were purified and conjugated with DOTA for (64)Cu labeling. The DOTA conjugates were tested in vitro for VEGFR-2 specificity and functional activity. In vivo tumor targeting efficacy and pharmacokinetics of (64)Cu-labeled VEGF(121) and VEGF(DEE) were compared using an orthotopic 4T1 murine breast tumor model. Blocking experiments, biodistribution studies, and immunofluorescence staining were carried out to confirm the noninvasive imaging results.

RESULTS

Cell binding assay demonstrated that VEGF(DEE) had about 20-fold lower VEGFR-1 binding affinity and only slightly lower VEGFR-2 binding affinity as compared with VEGF(121). MicroPET imaging studies revealed that both (64)Cu-DOTA-VEGF(121) and (64)Cu-DOTA-VEGF(DEE) had rapid and prominent activity accumulation in VEGFR-2-expressing 4T1 tumors. The renal uptake of (64)Cu-DOTA-VEGF(DEE) was significantly lower than that of (64)Cu-DOTA-VEGF(121) as rodent kidneys expressed high levels of VEGFR-1 based on immunofluorescence staining. Blocking experiments and biodistribution studies confirmed the VEGFR specificity of (64)Cu-DOTA-VEGF(DEE).

CONCLUSIONS

We have developed a VEGFR-2-specific PET tracer, (64)Cu-DOTA-VEGF(DEE). It has comparable tumor targeting efficacy to (64)Cu-DOTA-VEGF(121) but much reduced renal toxicity. This tracer may be translated into the clinic for imaging tumor angiogenesis and monitoring antiangiogenic treatment efficacy.

OBJECTIVE

Both vascular endothelial growth factor (VEGF) and transforming growth factor-beta1 (TGF-beta1) are expressed in higher than normal concentrations in the penumbra of patients after ischemic stroke. Because both cytokines are central to the processes of angiogenesis, tissue inflammation, and fibrosis, we performed serial measurements of these cytokines in patients with cerebral infarction and determined their relationship to stroke etiology and volume.

METHODS

We serially (at days 0, 1, 3, 7, and 14) measured the serum levels of VEGF and active TGF-beta1 in 29 patients with acute ischemic stroke. Age-matched healthy subjects (n=26) were used as controls.

RESULTS

Expression of VEGF was significantly increased in the majority of patients after acute stroke at each of the time points compared with normal controls. Highest expression occurred at day 7 (588+/-121 pg/mL; P=0.005), and it remained significantly elevated at 14 days after stroke. Expression of VEGF correlated with infarct volume, clinical disability (Scandinavian Stroke Scale), and peripheral leukocytosis and was significantly higher in patients with atherothrombotic large-vessel disease and ischemic heart disease (P<0.05 in all cases). In contrast, expression of active TGF-beta1 was not significantly different from control patients at any of the measured time points. When the mean concentration of TGF-beta1 from each patient (pooled time points) was compared with the control mean, a significant increase was found in only 2 patients, whereas levels decreased in 12 patients (P<0.05). There was no correlation between circulating active TGF-beta1 and VEGF expression, leukocytosis, stroke subtype, or patient disability as assessed by Scandinavian Stroke Scale score.

CONCLUSIONS

VEGF but not TGF-beta1 showed a dramatic increase in serum of stroke patients. Correlation between stroke severity and VEGF concentration suggests it could be involved in the subsequent repair processes resulting in partial recovery after stroke. Correlation between VEGF expression and peripheral leukocytosis suggests that these changes may also reflect the immunologic status of the patient. VEGF may play an important role in the pathophysiology of acute ischemic stroke and could be of value in future treatment strategies.

The usefulness of chemotherapy in patients with stage II disease continues to be debated. Biological prognostic <em>factors</em> may allow further insight into the optimal treatment strategy for patients with node-negative disease. <em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) seems to be essential for angiogenesis and for the <em>growth</em> of colorectal cancer. Recently, it was shown able to predict disease recurrence in patients with stage II colon cancer. Specimens of surgically resected colon cancer were immunostained for VEGF. Consecutive patients referred to the study institutions were considered eligible for this study. The main inclusion criteria were stage II tumor, sufficient tumor material, and adequate follow-up information. Analysis was performed on <em>121</em> patients. The recurrence rate in the patients with VEGF-positive tumors was 50% (18 of 36 patients), which was significantly higher than that observed in patients with VEGF-negative tumors [11.7% (10 of 85 patients); P = 0.001]. Also the degree of VEGF immunoreactivity was significantly higher in 28 relapsing patients compared with 93 disease-free patients (mean VEGF score, 2.84 0.38 versus 0.66 +/- 0.17; P = 0.0001). VEGF may be used in a clinical setting to identify patients at high risk for relapse who may benefit from adjuvant treatment including new therapeutic strategies such as monoclonal antibody neutralizing VEGF.

To assess the possible involvement of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) in the pathology of osteoarthritic (OA) cartilage, we examined the expression of VEGF isoforms and their receptors in the articular cartilage, and the effects of VEGF on the production of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in OA chondrocytes. Reverse transcriptase-polymerase chain reaction analyses demonstrated that mRNAs for three VEGF isoforms (VEGF(<em>121</em>), VEGF(165), and VEGF(189)) are detectable in all of the OA and normal (NOR) cartilage samples. However, the mRNA expression of their receptors (VEGFR-1 = Flt-1, VEGFR-2 = KDR and neuropilin-1) was recognized only in the OA samples. The protein expression of VEGFR-1 and VEGFR-2 in OA chondrocytes was also demonstrated by immunohistochemistry of the OA cartilage tissue and cultured OA chondrocytes. In situ hybridization and immunohistochemistry indicated that VEGF is expressed in the chondrocytes in the superficial and transitional zones of OA cartilage. A linear correlation was obtained between VEGF immunoreactivity and Mankin scores in the cartilage (r = 0.906, P < 0.001). The production levels of VEGF determined by enzyme-linked immunosorbent assay were significantly 3.3-fold higher in OA than in NOR samples (P < 0.001). Among MMP-1, -2, -3, -7, -8, -9, and -13, TIMP-1 and -2 measured by their sandwich enzyme immunoassay systems, the production of MMP-1 and MMP-3 but not TIMP-1 or TIMP-2 was significantly enhanced by the treatment of cultured OA chondrocytes with VEGF (P < 0.05), whereas no such effect was obtained with cultured NOR chondrocytes. These results demonstrate that VEGF and its receptors are expressed in OA cartilage, and suggest the possibility that VEGF is implicated for the destruction of OA articular cartilage through the increased production of MMPs.

OBJECTIVE

Vascular endothelial growth factor (VEGF), a potent angiogenic mediator, can be delivered to targeted tissues by means of a replication-deficient adenovirus (Ad) vector. We hypothesized that direct administration of Ad vector expressing the VEGF121 complementary deoxyribonucleic acid (AdGVVEGF121.10) into regions of ischemic myocardium would enhance collateral vessel formation and improve regional perfusion and function.

METHODS

Yorkshire swine underwent thoracotomy and placement of an Ameroid constrictor (Research Instruments & MFG, Corvallis, Ore.) on the circumflex coronary artery. Three weeks later, myocardial perfusion and function were assessed by single photon emission computed tomography imaging (SPECT) with 99mTc-labeled sestamibi and by echocardiography during rest and stress. AdGVVEGF121.10 (n = 7) or the control vector, AdNull (n = 8), was administered directly into the myocardium at 10 sites in the circumflex distribution (10(8) pfu/site). Four weeks later, these studies were repeated and ex vivo angiography was performed.

RESULTS

SPECT imaging 4 weeks after vector administration demonstrated significant reduction in the ischemic area at stress in AdGVVEFG121.10-treated animals compared with AdNull control animals (p = 0.005). Stress echocardiography at the same time demonstrated improved segmental wall thickening in AdGVVEGF121.10 animals compared with AdNull control animals (p = 0.03), with AdGVVEGF121.10 animals showing nearly normalized function in the circumflex distribution. Collateral vessel development assessed by angiography was also significantly greater in AdGVVEGF121.10 animals than in AdNull control animals (p = 0.04), with almost complete reconstitution of circumflex filling in AdGVVEGF121.10 animals.

CONCLUSIONS

An Ad vector expressing the VEGF121 cDNA induces collateral vessel development in ischemic myocardium and results in significant improvement in both myocardial perfusion and function. Such a strategy may be useful in patients with ischemic heart disease in whom complete revascularization is not possible.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> A (VEGFA) and the type III receptor tyrosine kinase receptors (RTKs) are both required for the differentiation of <em>endothelial</em> cells (vasculogenesis) and for the sprouting of new capillaries (angiogenesis). We have isolated a duplicated zebrafish VegfA locus, termed VegfAb, and a duplicate RTK locus with homology to KDR/FLK1 (named Kdrb). Morpholino-disrupted VegfAb embryos develop a normal circulatory system until approximately 2 to 3 days after fertilization (dpf), when defects in angiogenesis permit blood to extravasate into many tissues. Unlike the VegfAa(<em>121</em>) and VegfAa(165) isoforms, the VegfAb isoforms VegfAb(171) and VegfAb(210) are not normally secreted when expressed in mammalian tissue culture cells. The Kdrb locus encodes a 1361-amino acid transmembrane receptor with strong homology to mammalian KDR. Combined knockdown of both RTKs leads to defects in <em>vascular</em> development, suggesting that they cooperate in mediating the <em>vascular</em> effects of VegfA in zebrafish development. Both VegfAa and VegfAb can individually bind and promote phosphorylation of both Flk1 (Kdra) and Kdrb proteins in vitro. Taken together, our data support a model in the zebrafish, in which duplicated VegfA and multiple type III RTKs mediate <em>vascular</em> development.

Four <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) splice variants containing <em>121</em>, 165, 189, and 206 amino acids are produced from a single human gene as a result of alternative splicing. VEGF<em>121</em> is not a heparin-binding protein, while the other VEGF species possess heparin binding ability. YU-ZAZ6 human melanoma cells expressed the mRNA encoding the VEGF receptor flt-1, but not the mRNA encoding the VEGF receptor KDR/flk-1. Both VEGF<em>121</em> and VEGF165 bound to the VEGF receptors of these cells. Unexpectedly, heparin inhibited the binding of VEGF<em>121</em> as well as the binding of VEGF165 to the VEGF receptors of the melanoma cells. Digestion of the cells with heparinase also inhibited the binding of both VEGF variants. The VEGF165 binding ability of heparinase-digested cells could be partially restored by the addition of exogenous heparin to the binding reaction. In contrast, the addition of heparin to heparinase-digested cells did not restore VEGF<em>121</em> binding. These results suggest that cell-surface heparan sulfates may regulate the binding ability of the VEGF receptors of the melanoma cells. They also indicate that heparin is not able to fully substitute for cell surface-associated heparan sulfates since VEGF<em>121</em> binding to the VEGF receptors of heparinase-treated cells is not restored by heparin. These data suggest that changes in the composition of cell-surface heparin-like molecules may differentially affect the interaction of various VEGF isoforms with VEGF receptors.

Extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) is thought to promote tumor angiogenesis mostly through its protease-inducing function and more recently by its ability to increase tumor cell expression of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF). In this study, we present evidence that EMMPRIN can promote angiogenesis by a direct effect on <em>endothelial</em> cells through a paracrine regulation of the VEGF/VEGF-receptor (VEGFR) system. Using human micro<em>vascular</em> <em>endothelial</em> cell line-1 <em>endothelial</em> cells, we show that EMMPRIN selectively increased the soluble VEGF isoforms (<em>121</em> and 165), but not the matrix-bound VEGF 189 form. In addition, EMMPRIN up-regulated the expression of VEGFR-2 without an effect on VEGFR-1. This increase in VEGFR-2 was responsible for the observed EMMPRIN stimulation of the migratory and tube formation capacity of <em>endothelial</em> cells. EMMPRIN's effects, which were matrix metalloproteinase and urokinase-type plasminogen activator independent, were mediated primarily through hypoxia-inducible <em>factor</em>-2alpha expression, also up-regulated by EMMPRIN. VEGFR-2 increase was also observed in vivo in a mouse model of xenograph tumors overexpressing EMMPRIN. These results suggest that in addition to increasing protease production, EMMPRIN may contribute to the formation of a reactive stroma also through the up-regulation of hypoxia-inducible <em>factor</em>-2alpha, VEGFR-2, and the soluble forms of VEGF in <em>endothelial</em> cells, thus directly regulating the angiogenic process.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is known to occur as at least six differentially spliced variants, giving rise to mature isoforms containing <em>121</em>, 145, 165, 183, 189 and 206 amino acids. However, little is yet known concerning the in vivo activities of this differential splicing. Stably transfected MCF-7 breast carcinoma cells were constructed that secreted comparable amounts of the <em>121</em>, 165 or 189 isoforms. Rabbit corneal angiogenesis assays showed the VEGF<em>121</em> transfectant to have much greater angiogenic activity than the 165 or 189 expressing MCF-7 cells. While the VEGF<em>121</em>-expressing MCF-7 cells were reproducibly more tumorigenic than the control transfectants, this was not the case with the VEGF165- or VEGF189-expressing cells. More surprising was the observation that VEGF189 located to the nucleus, consistent with the presence of a highly conserved nuclear localization sequence in exon 6a that is expressed in VEGF189 but not <em>121</em> or 165. It was concluded that the VEGF<em>121</em> isoform is both more angiogenic and tumorigenic than are the 165 and 189 isoforms. This is probably due to the ability of the <em>121</em> isoform, unlike the 165 and 189 isoforms, to freely diffuse from the cells producing it.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is a pleiotropic polypeptide that mediates <em>endothelial</em>-cell-specific responses such as induction of proliferation and <em>vascular</em> leakage. We examined the expression of VEGF messenger RNA (mRNA) and protein by human eosinophils in response to granulocyte-macrophage colony-stimulating <em>factor</em> (GM-CSF) and interleukin-5 (IL-5). Immunoreactive VEGF protein was detected in freshly isolated eosinophils by immunocytochemistry. Eosinophils spontaneously released VEGF protein in culture medium, and this release was upregulated by GM-CSF or IL-5. Freshly isolated eosinophils constitutively expressed VEGF mRNA. Although incubation of eosinophils in culture medium reduced steady-state VEGF mRNA levels, eosinophil VEGF mRNA levels were enhanced by GM-CSF and IL-5, and this enhancement was blocked by the transcription inhibitor actinomycin D. Analysis of alternatively spliced mRNA species revealed that eosinophils contained transcripts mainly encoding for the <em>121</em>- and 165-amino-acid forms of VEGF. VEGF mRNA expression and VEGF release in cytokine-stimulated eosinophils were significantly reduced by treatment with a glucocorticosteroid, a protein-tyrosine kinase inhibitor, or a protein kinase C inhibitor. Cytokine-activated eosinophils may be an important source of a <em>vascular</em> permeability <em>factor</em>, namely VEGF, thus contributing to tissue edema formation at sites of allergic inflammation.

Tumors express more than a single angiogenic <em>growth</em> <em>factor</em>. To investigate the relative impact of fibroblast <em>growth</em> <em>factor</em>-2 (FGF-2) and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) on tumor <em>growth</em> and neo<em>vascular</em>ization, we generated tumor cell transfectants differing for VEGF and/or FGF-2 expression. Human endometrial adenocarcinoma HEC-1-B-derived Tet-FGF-2 cells that express FGF-2 under the control of the tetracycline-responsive promoter (Tet-off system) were further transfected with a VEGF(<em>121</em>) anti-sense (AS-VEGF) cDNA. Next, Tet-FGF-2 and AS-VEGF/Tet-FGF-2 cells were transplanted subcutaneously in nude mice that received tetracycline or not in the drinking water. Simultaneous expression of FGF-2 and VEGF in Tet-FGF-2 cells resulted in fast-<em>growing</em> lesions characterized by high blood vessel density, patency and permeability, and limited necrosis. Blood vessels were highly heterogeneous in size and frequently associated with pericytes. Inhibition of FGF-2 production by tetracycline caused a significant decrease in tumor burden paralleled by a decrease in blood vessel density and size. AS-VEGF expression resulted in a similar reduction in blood vessel density associated with a significant decrease in pericyte organization, <em>vascular</em> patency, and permeability. The consequent decrease in tumor burden was paralleled by increased tumor hypoxia and necrosis. A limited additional inhibitory effect was exerted by simultaneous down-regulation of FGF-2 and VEGF expression. These findings demonstrate that FGF-2 and VEGF stimulate <em>vascular</em>ization synergistically but with distinctive effects on vessel functionality and tumor survival. Blockade of either one of the two <em>growth</em> <em>factors</em> results in a decrease in blood vessel density and, consequently, in tumor burden. However, inhibition of the expression of VEGF, but not of FGF-2, affects also vessel maturation and functionality, leading to tumor hypoxia and necrosis. Our experimental model represents an unique tool to investigate anti-neoplastic therapies in different angiogenic environments.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> A (VEGF-A) is a potent inducer of angiogenesis. We now show that VEGF-A-induced adhesion and migration of human <em>endothelial</em> cells are dependent on the integrin alpha9beta1 and that VEGF-A is a direct ligand for this integrin. Adhesion and migration of these cells on the 165 and <em>121</em> isoforms of VEGF-A depend on cooperative input from alpha9beta1 and the cognate receptor for VEGF-A, VEGF receptor 2 (VEGF-R2). Unlike alpha3beta1or alphavbeta3 integrins, alpha9beta1 was also found to bind the <em>121</em> isoform of VEGF-A. This interaction appears to be biologically significant, because alpha9beta1-blocking antibody dramatically and specifically inhibited angiogenesis induced by VEGF-A165 or -<em>121</em>. Together with our previous findings that alpha9beta1 directly binds to VEGF-C and -D and contributes to lymphangiogenesis, these results identify the integrin alpha9beta1 as a potential pharmacotherapeutic target for inhibition of pathogenic angiogenesis and lymphangiogenesis.

Administration of adenovirus (Ad) vectors to immunologically naive experimental animals almost invariably results in the induction of systemic anti-Ad neutralizing antibodies. To determine if the human systemic humoral host responses to Ad vectors follow a similar pattern, we evaluated the systemic (serum) anti-Ad serotype 5 (Ad5) neutralizing antibodies in humans after administration of first generation (E1(-) E3(-)) Ad5-based gene transfer vectors to different hosts. AdGVCFTR.10 (carrying the normal human cystic fibrosis [CF] transmembrane regulator cDNA) was sprayed (8 x 10(7) to 2 x 10(10) particle units [PU]) repetitively (every 3 months or every 2 weeks) to the airway epithelium of 15 individuals with CF. AdGVCD.10 (carrying the Escherichia coli cytosine deaminase gene) was administered (8 x 10(8) to 8 x 10(9) PU; once a week, twice) directly to liver metastasis of five individuals with colon cancer and by the intradermal route (8 x 10(7) to 8 x 10(9) PU, single administration) to six healthy individuals. AdGVVEGF<em>121</em>.10 (carrying the human <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> <em>121</em> cDNA) was administered (4 x 10(8) to 4 x 10(9.5) PU, single administration) directly to the myocardium of 11 individuals with ischemic heart disease. Ad vector administration to the airways of individuals with CF evoked no or minimal serum neutralizing antibodies, even with repetitive administration. In contrast, intratumor administration of an Ad vector to individuals with metastatic colon cancer resulted in a robust antibody response, with anti-Ad neutralizing antibody titers of 10(2) to >10(4). Healthy individuals responded to single intradermal Ad vector variably, from induction of no neutralizing anti-Ad antibodies to titers of 5 x 10(3). Likewise, individuals with ischemic heart disease had a variable response to single intramyocardial vector administration, ranging from minimal neutralizing antibody levels to titers of 10(4). Evaluation of the data from all trials showed no correlation between the peak serum neutralizing anti-Ad response and the dose of Ad vector administered (P>> 0.1, all comparisons). In contrast, there was a striking correlation between the peak anti-Ad5 neutralizing antibody levels evoked by vector administration and the level of preexisting anti-Ad5 antibodies (P = 0.0001). Thus, unlike the case for experimental animals, administration of Ad vectors to humans does not invariably evoke a systemic anti-Ad neutralizing antibody response. In humans, the extent of the response is dictated by preexisting antibody titers and modified by route of administration but is not dose dependent. Since the extent of anti-Ad neutralizing antibodies will likely modify the efficacy of administration of Ad vectors, these observations are of fundamental importance in designing human gene therapy trials and in interpreting the efficacy of Ad vector-mediated gene transfer.

OBJECTIVE

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) induces angiogenesis and <em>vascular</em> permeability and is thought to be operative in several ocular <em>vascular</em> diseases. The VEGF isoforms are highly conserved among species; however, little is known about their differential biological functions in adult tissue. In the current study, the inflammatory potential of two prevalent VEGF isoform splice variants, VEGF(120(<em>121</em>)) and VEGF(164(165)), was studied in the transparent and a<em>vascular</em> adult mouse cornea.

METHODS

Controlled-release pellets containing equimolar amounts of VEGF(120) and VEGF(164) were implanted in corneas. The mechanisms underlying this differential response of VEGF isoforms were explored. The response of VEGF in cultured endothelial cells was determined by Western blot analysis. The response of VEGF isoforms in leukocytes was also investigated.

RESULTS

VEGF(164) was found to be significantly more potent at inducing inflammation. In vivo blockade of VEGF receptor (VEGFR)-1 significantly suppressed VEGF(164)-induced corneal inflammation. In vitro, VEGF(165) more potently stimulated intracellular adhesion molecule (ICAM)-1 expression on endothelial cells, an effect that was mediated by VEGFR2. VEGF(164) was also more potent at inducing the chemotaxis of monocytes, an effect that was mediated by VEGFR1. In an immortalized human leukocyte cell line, VEGF(165) was found to induce tyrosine phosphorylation of VEGFR1 more efficiently.

CONCLUSIONS

Taken together, these data identify VEGF(164(165)) as a proinflammatory isoform and identify multiple mechanisms underlying its proinflammatory biology.

The effects of exogenous <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) on angiogenesis, blood-brain barrier permeability and astroglial proliferation in the adult rat CNS in situ were investigated. Recombinant human VEGF(165) (25 or 50 ng/ml) was delivered for up to 1 week using either intracerebral osmotic minipumps or less traumatic subdural gelatin sponge placement. By 3 days, VEGF delivery caused significantly increased cerebral angiogenesis (25 ng/ml was most effective) in both experimental models when compared to saline controls; VEGF infusion resulted in a 100% increase in an index of <em>vascular</em> proliferation, and gelatin sponge delivery produced a 65% increase. The blood-brain barrier hallmark <em>endothelial</em> glucose transporter-1 was not present in nascent <em>vascular</em> sprouts. Infusion of VEGF produced extensive protein leakage that persisted after saline-induced permeability was mostly resolved, while gelatin sponge administration caused milder barrier dysfunction. Administration of the angiogenic <em>factor</em> had unexpected proliferative effects on astroglia in both models, resulting in an 80-85% increase in mitotically active astroglia when compared to controls. Immunohistochemical results and semi-quantitative reverse transcriptase-polymerase chain reaction indicated that the VEGF receptors flk-1 and flt-1 were up-regulated in response to the infusion trauma; flt-1 was localized to reactive astroglia, while flk-1 was expressed in <em>vascular</em> endothelium but predominantly in neuronal somata and processes adjacent to the delivery site. mRNA for the VEGF(<em>121</em>), VEGF(165) and VEGF(188) isoforms was also increased after delivery of the recombinant protein. These data show that VEGF application has substantial proliferative effects on CNS endothelium and astroglia and causes up-regulation of its own message. Flt-1 and flk-1 receptor mRNAs and proteins are up-regulated in both <em>vascular</em> and non-<em>vascular</em> cell types following infusion trauma. From these results we suggest that administered VEGF has heretofore unanticipated pleiotrophic effects in the adult CNS.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> A (VEGF-A) and its receptors, Flt-1/FLT-1 (VEGFR-1) and Flk-1/KDR (VEGFR-2), are key regulators of tumor angiogenesis and tumor <em>growth</em>. The purpose of this study was to determine the antiangiogenic and antitumor efficacies of a vasculature-targeting fusion toxin (VEGF(<em>121</em>)/rGel) composed of the VEGF-A isoform VEGF(<em>121</em>) linked with a G(4)S tether to recombinant plant toxin gelonin (rGel) in an orthotopic glioblastoma mouse model by use of noninvasive in vivo bioluminescence imaging (BLI), MRI, and PET.

METHODS

Tumor-bearing mice were randomized into 2 groups and balanced according to BLI and MRI signals. PET with (64)Cu-1,4,7,10-tetraazacyclododedane-N,N',N'',N'''-tetraacetic acid (DOTA)-VEGF(<em>121</em>)/rGel was performed before VEGF(<em>121</em>)/rGel treatment. (18)F-Fluorothymidine ((18)F-FLT) scans were obtained before and after treatment to evaluate VEGF(<em>121</em>)/rGel therapeutic efficacy. In vivo results were confirmed with ex vivo histologic and immunohistochemical analyses.

RESULTS

Logarithmic transformation of peak BLI tumor signal intensity revealed a strong correlation with MRI tumor volume (r = 0.89, n = 14). PET with (64)Cu-DOTA-VEGF(<em>121</em>)/rGel before treatment revealed a tumor accumulation (mean +/- SD) of 11.8 +/- 2.3 percentage injected dose per gram at 18 h after injection, and the receptor specificity of the tumor accumulation was confirmed by successful blocking of the uptake in the presence of an excess amount of VEGF(<em>121</em>). PET with (18)F-FLT revealed significant a decrease in tumor proliferation in VEGF(<em>121</em>)/rGel-treated mice compared with control mice. Histologic analysis revealed specific tumor neovasculature damage after treatment with 4 doses of VEGF(<em>121</em>)/rGel; this damage was accompanied by a significant decrease in peak BLI tumor signal intensity.

CONCLUSIONS

The results of this study suggest that future clinical multimodality imaging and therapy with VEGF(<em>121</em>)/rGel may provide an effective means to prospectively identify patients who will benefit from VEGF(<em>121</em>)/rGel therapy and then stratify, personalize, and monitor treatment to obtain optimal survival outcomes.

Angiogenesis has been linked to increased metastasis formation and decreased overall survival in patients with various tumors, including and neck squamous cell carcinomas (HNSCC). <em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is a key regulator of angiogenesis. In the present study, we evaluated VEGF expression and microvessel density (MVD), a quantitative means of angiogenesis, in both experimental and clinical models of HNSCC. Analysis of VEGF RNA expression in cell lines of keratinocyte origin [HNSCC, facial skin squamous cell carcinoma (SCC), and transformed but nontumorigenic keratinocytes] and normal skin keratinocytes revealed two VEGF transcripts corresponding to proteins of 165 and <em>121</em> amino acids in length, with the transcript for the 165-amino acid species predominating. Six of eight SCC cell lines showed increased levels of one or both transcripts, and seven SCC cell lines and the transformed keratinocyte cell line showed increased protein expression. We then evaluated VEGF protein expression in human head and neck specimens containing normal epithelium (n = 10), dysplasia or carcinoma in situ (CIS; n = 15), early invasive SCCs (n = 9), advanced primary SCCs (n = 10), lymph node metastases (n = 3), and s.c. tumors or cysts (n = 7) formed in severe combined immunodeficient mice. Intense VEGF staining was found in the majority of advanced primary SCCs, lymph node metastases, and human SCCs in severe combined immunodeficient mice, whereas no dysplasia, CIS, or early SCCs showed intense immunostain. A highly significant increase (P = 0.0001) in VEGF expression was seen in the advanced SCC versus dysplasias and CIS lesions, as was the difference between SCC versus normal epithelium from nonsmokers (P = 0.01). VEGF expression in advanced primary cancers was greater (P = 0.002) and, in early cancers, marginally greater (P = 0.05) than adjacent normal mucosa. MVD increased with the progression of preinvasive disease (P = 0.04). VEGF expression and MVD (both, P = 0.003) were directly associated with tumor aggressiveness in experimental tumors. These findings suggest a role for VEGF in both clinical and experimental HNSCC.

BACKGROUND

Vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 and -2 are considered to play a major in tumor angiogenesis, which is a prerequisite for growth of solid tumors. Glioblastoma multiforme is a prominent example of VEGF-induced tumor vascularization; however, little is known about VEGF and in particular VEGFR expression in other types of brain tumors.

METHODS

VEGFR mRNA was quantified by real time RT-PCR in 12 different types of brain tumors and compared to VEGF protein content measured by ELISA. VEGF splice variants were determined by an RT-PCR method.

RESULTS

VEGF protein was highest in glioblastoma and metastatic kidney tumors. In all types of tumors the diffusible splice forms VEGF(121) and VEGF(165) were expressed; VEGF(189) was minor in a few tumors. Expression of VEGF receptors did not necessarily correlate with VEGF content. Both were highly expressed in glioblastomas, but in meningiomas VEGF was low and VEGFR high, and in metastatic tumors the reverse. With few exceptions, in particular oligodendrogliomas, VEGFR-1 expression was parallel to VEGFR-2 expression. Interestingly, for the astrocytic gliomas, the expression of VEGFR correlated well to the tumor malignancy, even better than VEGF content.

CONCLUSIONS

These results show that VEGF and VEGFR expression in various types of brain tumors differ and are not necessarily parallel.